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PubMed2002

The terminal proteins of linear Streptomyces chromosomes and plasmids: a novel class of replication priming proteins.

The chromosomes of the soil bacteria Streptomyces, unlike those of most other bacteria, are linear DNA molecules. Their telomeres contain long-terminal inverted repeats and covalently bound terminal proteins (TPs). These bacteria also harbour linear plasmids that share the same structural features. In this study, we demonstrated that the TP was covalently bound to the 5' ends as proposed previously. A linear plasmid with chromosomal telomeres was constructed and used to purify the TPs of the Streptomyces coelicolor A3(2) chromosome. A 20 kDa protein and its 10 kDa degradation product were isolated and their sequences determined by mass spectrometry. The coding sequence (tpgC) was about 100 kb from the right end of the chromosome. Two tpg homologues were identified by sequencing the 50kb linear plasmid SLP2 of Streptomyces lividans: tpgSLP2 at 6 kb from the left end and a putative tpg pseudogene at 8 kb from the right. The latter was in a terminal repeat shared by the right end of SLP2 and both ends of the S. lividans chromosome. The lack of the typical Streptomyces codon preference in this open reading frame suggests that it is a pseudogene. The close physical linkage between the tpg genes and their cognate telomeres would favour their cosegregation and co-evolution. All the Tpg polypeptides are similar in length (184-185 amino acids) and sequences, which include a putative helix domain that is homologous to part of the DNA-binding 'thumb' domain of HIV reverse transcriptase, and a putative amphiphilic beta-sheet that may be involved in the observed self-aggregation of the TP and/or the proposed membrane binding.

Amino Acid Sequence↗
PubMed1975

Transition of R factor NR1 in Proteus mirabilis: molecular structure and replication of NR1 deoxyribonucleic acid.

The structure of R factor NR1 DNA in Proteus mirabilis has been studied by using the techniques of CsCl density gradient centrifugation, sedimentation in neutral and alkaline sucrose gradients, and electron microscopy. It has been shown that the nontransitioned form of NR1 DNA isolated from P. mirabilis cultured in drug-free medium is a37-mum circular deoxyribonucleic acid (DNA) with a density of 1.712 g/ml in a neutral CsCl gradient. This circular molecule is a composite structure consisting of a 29-mum resistance transfer factor containing the tetracycline-resistance genes (RTF-TC) and an 8-mum r-determinants component conferring resistance to chloramphenicol (CM), streptomycin/spectinomycin, and the sulfonamides. There are one to two copies of NR1 per chromosome equivalent of DNA in exponential-phase cells cultured in Penassay broth. After growth of PM15/NR1 in medium containing 100 mug of CM per ml, the density of the NR1 DNA increased from 1.712 g/ml to approximately 1.718 g/ml and the proportion of NR1 DNA relative to the chromosome is amplified about 10-fold. The changes in R factor DNA structure which accompany this phenomenon (termed the transition) have been studied. DNA density profiles of the transitioned NR1 DNA consist of a 1.718 g/ml band which is skewed toward the less dense side. The transitioned NR1 DNA consists of molecules containing the RTF-TC element attached to multiple copies of r-determinants DNA (poly-r-determinant R factors) and multimeric and monomeric autonomous r-determinants structures. Poly-r-determinant R factors have a density intermediate between the basic composite structure (1.712 g/ml) and r-determinants DNA (1.718 g/ml). These species presumably account for the skewing of the 1.718-g/ml DNA band toward the less dense side. When transitioned cells are subsequently cultured in drug-free medium, poly-r-determinant R factors and autonomous poly-r-determinants undergo dissociation to form smaller structures containing fewer copies of r-determinants. This process continues until, after prolonged growth in drug-free medium the NR1 DNA returns to the nontransitioned state which consists of an RTF-TC and a single copy of r-determinants.

Chloramphenicol↗
PubMed1990

The AT richness and gid transcription determine the left border of the replication origin of the E. coli chromosome.

We have identified novel, cis-acting elements which enhance in vivo the replication activity of plasmids carrying the minimal oriC of Escherichia coli. These are (i) the AT rich sequence ('AT-cluster') which exists immediately left of the 13mer repeats and (ii) the gid transcriptional unit. The 'AT-cluster' was functionally replaced by an unrelated AT rich sequence. This was also the case for the left and middle 13mers; they were substituted by the AT rich fragment from mini-F plasmid. The left 13mer was replaced by the AT rich sequence which did not show the 'reduced helical stability' known as the important character of the 13mer region. In contrast to these results, the right 13mer sequence was strictly required. As to the effect of the transcription from the gid promoter, the minimal oriC was activated only when the transcription was directed away from the left side of it. mioC transcription proceeding toward the oriC had no effect on the activation. Mutations in the DnaA boxes were partially suppressed by gid transcription leaving oriC from the left side. From these results, we propose that the AT richness is a determinant to identify the left border of oriC. It is presumed that gid transcription introduces negative superhelicity at the AT rich region and facilitates DnaA dependent duplex opening.

Adenine↗
PubMed1988

Coordination of chromosome replication initiation in Escherichia coli: effects of different dnaA alleles.

The synchrony of initiation of chromosomal replication in single cells was determined in ten different dnaA(Ts) mutants. After inhibiting the initiation of replication but allowing initiated rounds of replication to terminate, we measured the number of fully replicated chromosomes per individual cell by flow cytometry. Synchronous initiation at the several independent origins (oriC) in single rapidly growing cells would give 2'' (n = 0,1,2,3,...) chromosomes per cell, whereas asynchronous initiation was indicated by the presence of a different number of chromosomes. Mutations mapping in the central part of the dnaA gene (dnaA5, dnaA46, dnaA601, dnaA602, and dnaA604) lead to a high degree of asynchrony (class I mutants), whereas mutations mapping in either of the distal parts of the gene (dnaA508, dnaA167, dnaA203, and dnaA204) yielded a low degree of asynchrony at the permissive temperature (class 2 mutants). The dnaA205 mutant exhibited an intermediate degree of asynchrony. Mutants dnaA203 and dnaA204 (promoter distal) differed from the other class 2 mutants (dnaA167, dnaA508; promoter proximal) in that asynchrony increased no more than twofold between 25 and 37 degrees C compared with the more-than-fourfold increase in the latter. The high degree of asynchrony in class 1 mutants was independent of temperature and was not due to insufficient functional DnaA protein, because overproduction of DnaA46 protein did not decrease the asynchrony. The data demonstrate that the DnaA protein has functions in addition to acting positively in the initiation process and negatively as its own repressor, namely in coordinating initiations at all oriC sites within a single cell.

Alleles↗
PubMed1980

Bacterial growth studied by flow cytometry.

The feasibility of flow cytometry for measurements on bacteria has been demonstrated by measurements of DNA-associated fluorescence of Escherichia coli K-12 in various phases of cell growth. Bacteria were stained with a combination of ethidium bromide and mithramycin after fixation in 70% ethanol. Cultures grown to stationary phase accumulated in two peaks representing cells with two and four chromosomes. Qualitatively similar histograms were obtained with cells grown in the presence of chloramphenicol, whereas cells of the temperature sensitive strain E 177 (dnaA) ended up with only one chromosome per cell at the restrictive temperature. The fluorescence intensity of cells with one chromosome was about 10(3) times smaller than that of human diploid cells. Instrumental resolution at this level of intensity was CV = 5%, whereas peak widths corresponded to CV = 7-8%. Dyes bound to RNA did not appear to contribute significantly to the fluorescence.

Bacteriological Techniques↗
PubMed1979

Translocation of the pre-synaptic complex formed upon DNA uptake by Streptococcus sanguis and its inhibition by ethidium bromide.

Donor DNA in its initially bound, single-stranded form exists in a chromosomally-unassociated complex where it is resistant to exogenous DNase I but sensitive to micrococcal nuclease. Most of the complexes are readily recuperable from the supernatant of recipients converted into spheroplasts. Subsequent to formation of this superficially located complex, donor DNA progressively associates with the recipient chromosome into which it is eventually integrated. Treatment of recipients with ethidium bromide at various times after initial DNA binding almost immediately halts translocation of whatever donor material is not yet synapsed with the chromosome. On the other hand, donor DNA that has already synapsed experiences no difficulty in becoming genetically integrated. Some degradation occurs to DNA that fails to undergo translocation as a result of ethidium bromide treatment, the acid-soluble products appearing in the culture medium. DNA in untranslocated complexes surviving treatment is not appreciably different in single-strand length from that in untreated complexes. When these surviving complexes are isolated from a cell lysate, the contained DNA can be shown by spectrofluorometry to have bound the drug.

Chromosomes, Bacterial↗
PubMed1980

Coordinate expression of Escherichia coli dnaA and dnaN genes.

The defects of temperature-sensitive dnaA and dnaN mutants of Escherichia coli are complemented by a recombinant lambda phage, which carries the bacterial DNA segment composed of two EcoRI segments of 1.0 and 3.3 kilobases. Derivatives of the phage, which have an insertion segment of Tn3 in the dnaA gene, are much less active in expressing the dnaN gene function than the parent phage. The dnaN gene activity was determined as the efficiency of superinfecting phage to suppress loss of the viability of lambda lysogenic dnaN59 cells at the non-permissive temperature. Deletions that include the end of the dnaA gene distal to the dnaN gene also reduce the expression of the dnaN gene function. Deletion and insertion in the dnaN gene do not affect the expression of the dnaA gene function. The expression of the dnaN gene function by the dnaA- dnaN+ phages remains weak upon simultaneous infection with dnaA+ dnaN- phages. Thus the insertion and deletion of the dnaA gene influence in cis the expresion of the dnaN gene. We propose that the dnaA and dnaN genes constitute an operon, where the former is upstream to the latter.

Bacteriophage lambda↗
PubMed1980

Polyethyleneglycol-induced transformation of Bacillus subtilis protoplasts by bacterial chromosomal DNA.

Bacillus subtilis protoplasts, which in the presence of polyethyleneglycol (PEG) are transformed by plasmid DNA (Chang and Cohen 1979) can also be transformed under these conditions by chromosomal DNA. Transformation in this case occurs at a much lower frequency, not fully accounted for by the heterogeneity of this DNA. Another unexpected feature of the transformation studied, which may explain why it previously went unnoticed, is that DNA concentrations higher than 1--2 microgram/ml decrease the yield of transformants, without showing signs of general toxicity. PEG-induced protoplasts (PIP) transformation for chromosomal markers operates normally with protoplasts prepared from a non-transformable bacterial mutant. The evidence indicates that both native linear and plasmid DNAs must somehow be forced into the cells as a result of PEG action. Denatured chromosomal DNA however is almost inactive in PIP transformation. No competition between chromosomal and plasmid DNAs could be detected, when the DNA tested as inhibitor was in tenfold excess.

Bacillus subtilis↗
PubMed1979

DNA synthesis in UV-irradiated Escherichia coli K-12 strains carrying dnaA mutations.

It has been found that UV irradiation of dnaA mutants of E. coli K-12 enables the initiation of DNA synthesis at a temperature restrictive to these mutants. The UV-induced DNA synthesis is dependent on protein synthesis and on a transcriptional event at a time when protein synthesis is no longer required. In contrast to dnaA mutants UV irradiation fails to induce DNA synthesis in the two other initiation mutants dnaC2 and dnaB252. DNA synthesis at the restrictive temperature is initiated also when the tif-1 phenotype is expressed in the dnaA46 tif-1 double mutant. Possible mechanisms of the observed capability of dnaA mutants to synthesize DNA at the restrictive temperature after UV irradiation or under conditions of tif-1 expression are discussed.

Bacterial Proteins↗
PubMed1979

Transformation of Pseudomonas aeruginosa and Escherichia coli with resistance plasmid DNA: formation of smaller conjugative plasmids from RPI.

Sheared DNA from RPI, and R plasmid from a strain of Pseudomonas aeruginosa, was used to transform other strains of P. aeruginosa and Escherichia coli. From transformed cells other plasmids like RPI were isolated. These deletion plasmids were conjugally transferrable and confer resistance mainly against carbenicillin and tetracycline.

Carbenicillin↗
PubMed1977

Selective enrichment for genetic markers in DNA released by competent cultures of Bacillus subtilis.

Deoxyribonucleic acid is released into the growth medium by Bacillus subtilis at the time of competence. This DNA is enriched for the genetic markers which have previously been demonstrated to be elevated in membrane-DNA preparations and more recently in cell wall-DNA complexes. Furthermore, the purA16/leu-8 relative marker enrichment varies with time, reaching its highest point at the time of maximal competence. Enrichment remains elevated for at least 60 min further in the competence regimen. Thr results suggest that certain genetic markers of the B. subtilis chromosome are preferentially more available to the external medium as the development of competence proceeds.

Bacillus subtilis↗
PubMed1979

Tandem and inverted repeats of arginine genes in Escherichia coli: structural and evolutionary considerations.

Duplications of arg genes produced in the Rec+ and in the recA genetic backgrounds are shown by heteroduplex analysis to be strictly tandem at the level of resolution of this technique. The formation of these particular rearrangements therefore does not require the inclusion of transposons or other sequences of an appreciable size in their final structure. Duplications of short segments (about 2,000 nucleotides) appear unexpectedly stable when compared with duplications of longer segments (about 10,000 nucleotides). One of the structures analyzed displays two inversely repeated argE genes rearranged into an artificial divergent operon. The bearing of this observation on the origin of bipolar operons, of "mirror-image" map symmetries and on the production of inverted repeats in general, is discussed.

Arginine↗